title: Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression.
abstract: Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem-loop structure located downstream of the 3'splice site, and the other involves an exonic splicing silencer (ESS) situated 3' to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.
authors.affiliation: Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Huang
authors.initials: H
authors.lastname: Li
authors.forename: Zhijia
authors.initials: Z
authors.lastname: Wang
authors.forename: Xuexia
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuanming
authors.initials: Y
authors.lastname: Cheng
authors.forename: Zhiqin
authors.initials: Z
authors.lastname: Xie
authors.forename: James L
authors.initials: JL
authors.lastname: Manley
authors.forename: Ying
authors.initials: Y
authors.lastname: Feng
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D034622:RNA Interference; D012326:RNA Splicing / Q000502:physiology*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C492258:FUBP1 protein, human; D016601:RNA-Binding Proteins; D012313:RNA; D004265:DNA Helicases